The invention relates to a non-human transgenic animal in which the expression of at least one of the genes which code for opiate receptors is modified.
Opiatesxe2x80x94the prototype of which is morphinexe2x80x94are the most potent analgesics available to medicine today. However, their use is limited by a range of secondary effects, including effects on autonomous functions (constipation, respiratory depression, hypotension, diuresis) and psychotropic effects.
The range of actions of opiates is mediated by membrane receptors of the nervous system, which recognize and specifically bind these compounds. 20 years ago, these receptors were discovered by pharmacological studies. Three receptors have been identified: mu, delta and kappa receptors. The genes which code for these three receptors have been cloned and complementary DNA nucleotide sequences which code for the 3 receptors are shown in FIG. 11 (mu), FIG. 12 (delta) and FIG. 13 (kappa). Selective ligands of the three classes of receptors exist at present, and study of the action of these compounds suggests:
that the mu receptor is the privileged target of the prototype opiate morphine, which is the analgesic used the most for treatment of severe pain,
the mu receptor is also the main target of heroin, one of the most feared narcotics in the context of toxicomania,
the delta receptor is also said to be involved in control of pain and the emotional state (well-being), but to a lesser degree,
the kappa receptor, like the other two receptors, is said to play a role in the analgesic action of opiates. On the other hand, and in contrast to mu and delta, it is said to have a dysphorizing psychotropic action, an action which has been regarded as an advantage for the development of potent analgesics lacking a toxicomanogenic potential.
All the strategies devised in the last 20 years by the pharmaceuticals industries to develop an ideal analgesic are based on these pharmacological data. They comprise in vitro and in vivo analysis of the effect of opiate agonists or antagonists. The interpretation of the results is dependent on the mu/delta/kappa selectivity of the products studied and their pharmacokinetic properties for studies in vivo.
A range of pharmacological results seems to indicate the existence of several receptors in each of the classes mu, delta or kappa which could constitute distinct targets for the agonists of each of the classes of receptors. The question of whether only three receptors or several mu (xcexc), delta (xcex4) and kappa (xcexa) receptors exist has not been resolved at present.
Three genes which code for these receptors have been cloned very recently. Each of them corresponds to one of the above classes of receptors defined by pharmacology. Thus a mu gene, a delta gene and a kappa gene have been characterized at the molecular level. The involvement of these genes in the biological action of opiate substances in vivo has not been defined.
The genes which code for the opiate receptors have been cloned very recently (Kieffer B. (1995) Cellular and Molecular Neurobiology 15:615-635).
In the following, the gene of the xcexc receptor is called MOR, the gene of the 6 receptor is called DOR and the gene of the xcexa receptor is called KOR.
One of the objects of the invention is to provide an experimental model which enables targeting of medicaments which have potent analgesic properties without having the secondary effects of opiates of the morphine type.
One of the objects of the invention is to provide non-human transgenic mammalian animals in which at least one of the genes of the opiate receptors is no longer expressed.
One of the objects of the invention is to provide non-human transgenic mammalian animals in which the gene of the xcexc receptor is no longer expressed.
One of the objects of the invention is to provide non-human transgenic mammalian animals in which the gene of the xcex4 receptor is no longer expressed.
One of the objects of the invention is to provide non-human transgenic mammalian animals in which the gene of the xcexa receptor is no longer expressed.
One of the other objects of the invention is to provide an animal model which is capable of screening medicaments which act on pathologies involving at least one of the opiate receptors.
The invention relates to the use of a non-human transgenic mammalian animal in which the expression of at least one the genes which codes for the opiate receptors is modified, in particular suppressed in the tissues or cells of the brain, with respect to normal expression, in particular in the tissues or cells of the brain, for determination of a medicament which is active on pathologies involving the opiate receptors.
More precisely, the invention relates to the use of a non-human transgenic mammalian animal in which the expression of the gene which codes for an opiate receptor is modified, in particular in the nerve tissues, with respect to normal expression, in particular in the nerve tissues, for determination of a medicament which acts on pathologies involving the opiate receptors, in particular acute or chronic severe pain, toxicomania or the prevention or treatment of transplant rejections.
The term xe2x80x9cmammalianxe2x80x9d includes all mammals with the exception of humans, advantageously rodents, and in particular mice.
xe2x80x9cTransgenic animalxe2x80x9d is understood as meaning not only an animal in the genome of which an exogenous gene has been introduced, but also an animal in which expression of an endogenous gene has been deleted, either by interruption of the endogenous gene or by replacement of an endogenous gene or of a fragment thereof by a construction such that it no longer allows expression of the endogenous gene. Such animals will be called xe2x80x9cknock-outxe2x80x9d animals or those deficient in the said endogenous gene.
Normal expression of one of the opiate receptors can be defined by several methods:
1) Determination of the mRNA corresponding to one of the genes of the opiate receptors: this is possible by the technique of RNA transfer (Northern blot) in which the mRNAs are separated on denaturing agarose gel by electrophoresis; and the RNAs are then transferred and bound to a membrane of the nitrocellulose or nylon type. To reveal the presence of the RNAs corresponding to one of the genes of the opiate receptors, it is possible to use a probe corresponding to all or a fragment of the cDNA of the gene in question.
2) Determination of the amount of protein corresponding to one of the opiate receptors: this is possible by studying the bonding of an opiate ligand (agonist or antagonist), such as diprenorphin, which is non-selective with respect to opiate receptors, DAGO (selective with respect to xcexc), naltrindole (selective with respect to xcex4) and labelled CI977 (selective with respect to xcexa), to receptors present in a tissue (brain) homogenate. As regards the respective definitions of these ligands, these are shown in the legend of FIG. 2. In particular, the dissociation constant Kd of several specific ligands of one of the opiate receptors is known and is of the order of 1 nanomolar for the ligands generally used. It is furthermore known that the Bmax for the above ligands is of the order of 0.1 picomol/mg membrane protein for xcexc and xcex4 receptors and 0.02 picomol/mg membrane protein for the xcexa receptor in respect of the mouse brain. It is thus known that a saturation curve with this ligand on membrane extracts containing the three opiate receptors prepared from the brain and analysis of the results obtained from the saturation curves by the Scatchard method (determination of the number of receptor sites) should give Bmax affinity values divided by two in heterozygotes and zero Bmax values (not measurable) in homozygotes.
This therefore allows quantification of the amount corresponding to one of the opiate receptors.
According to one embodiment, the invention relates to the use of a non-human transgenic mammalian animal which no longer expresses the gene of the xcexc receptor or of the xcexa receptor or of the xcex4 receptor.
The modification and absence of expression of one of these genes can be determined in the following manner.
1) Mice where the gene of the opiate receptor has been modified such that it can no longer be expressed are first characterized in relation to the DNA by analysis by the DNA transfer method (Southern blot), with the aid of a probe consisting of a fragment containing all or some of the region of the opiate receptor, this probe being defined in the examples.
Hybridization of this probe clearly shows that the band corresponding to one of the opiate receptors of the wild type (that is to say normally present in animals) is no longer present in animals which are homozygous with respect to mutation, but is replaced by a band corresponding to the modified genome.
2) Analysis by RNA transfer (northern blot) of the RNA extracts of tissues which express one of the opiate receptors, in particular the brain, shows that there is no longer RNA expression corresponding to the wild gene. The probe used in this case is defined by the complementary DNA portion which codes for the xcexc opiate receptor of mice downstream of the unique BamHI site of the coding region up to the STOP codon.
3) Finally, it can also be demonstrated that the binding of specific ligands to one of the opiate receptors, in particular with the aid of the ligands DAGO (selective with respect to xcexc), naltrindole (selective with respect to xcex4) or CI977 (selective with respect to xcexa), is completely absent in these mice.
The tissues in which the expression of a gene which codes for an opiate receptor is modified are essentially the nerve tissues, and in particular the neuronal cells.
In the context of the invention, modification of the expression of the said gene in other types of cells, such as immune cells, is not excluded.
As regards the painful pathologies treated by the opiates to which the invention relates, there may be mentioned:
1. acute or chronic pain: pain due to excess nociception with tissue lesions, including cancer pain, postoperative pain, infectious pain and chronic pain of the inflammatory rheumatism and degenerative inflammatory rheumatism type,
2. chronic pain: pain due to lesion of the nervous system or neuropathic pain comprising (1) deafferentations (example: amputation) and deafferentations with nociception (example: postoperative residual lumbosciatalgia).
Furthermore, the opiate antagonists can have immuno-suppressant properties which can be used to benefit in the prevention or treatment of transplant rejection, but the mechanism of this biological action is unknown.
In this respect, naltrindole has a suppressant effect on the mixed lymphocyte reaction (MLR) in vitro and prevents transplant rejection in vivo (Arakawa, K., Akami, T., Okamoto, M., Nakai, I., Oka, T., Nagase, H. Transplantation proceedings (1993) 25:738-740). Experimental protocols regarding the study of transplant rejection are to be found in this reference. The transgenic mice of the invention enable the mu component to be evaluated (verses delta and kappa) in the response of the mice to drugs under development, for example compounds derived from naltrindole (delta antagonists) for development of an agent which blocks transplant rejection.
It has furthermore been shown that opiate alkaloids have an immunosuppressant activity on other immune functions: they block/inhibit the production of antibodies and killer activity (xe2x80x9cnatural killersxe2x80x9d), two essential components of immune responses against infections. Mice from which expression of opioid receptors has been deleted will therefore also be used as an animal model to test the action of immunosuppressant drugs of the opiate type developed for the above use for all the components of the immune response.
In the text above and below, xe2x80x9cxcexc opiate receptor genesxe2x80x9d are also understood as meaning the iso-forms generated by alternative splicing which are at the junction specific to the mu gene (between exon 3 and 4): Zimprich, A., Simon, T. and Hxc3x6lt, V. (1995) Cloning and expression of an isoform of the rat xcexc-opioid receptor (rMORIB) which differs in agonist-induced desensitizations from RMORI. FEBS 359:142-146 and Bare, L A., Mansson, E. and Yang, D M. (1994) Expressions of two variants of the human xcexc-opioid receptor mRNA in SK-N-SH cells and human brain. FEBS 354:213-216.
The invention also relates to the use of a non-human transgenic mammalian animal as described above which no longer expresses at least one of the following receptors: the opiate receptor of the mu type, the opiate receptor of the kappa type and the opiate receptor of the delta type.
The invention also relates to a non-human transgenic mammalian animal or mammalian cells containing the gene of the opiate receptor of the mu type in which a fragment of the gene of the receptor containing an exon, in particular exon 2, is
either replaced by all or part of a marker gene under the control of a suitable promoter,
or interrupted by the insertion between two contiguous nucleotides of all or part of a marker gene under the control of a suitable promoter, in particular the gene of resistance to neomycin (neo) under the control of the promoter phosphoglycerate kinase-1 (PGK-1), the expression of the gene of the mu type being suppressed.
According to an advantageous embodiment of the invention, the transgenic mammalian animal or the mammalian cells in which the xcexc gene is no longer expressed are such that they have an interruption in the xcexc for xcexcxcex4 and xcexa gene, in particular of an exon, and in particular exon 2, by the insertion between two contiguous nucleotides of all or part of a marker gene under the control of a suitable promoter, in particular the gene of resistance to neomycin (neo) under the control of the promoter phosphoglycerate kinase-1 (PGK-1), the expression of the gene of the mu type being suppressed.
The invention also relates to a non-human transgenic mammalian animal or mammalian cells containing the gene of the opiate receptor of the delta type in which a fragment of the gene of the receptor containing an exon, in particular exon 1, is
either replaced by all or part of a marker gene under the control of a suitable promoter,
or interrupted by the insertion between two contiguous nucleotides of all or part of a marker gene under the control of a suitable promoter, in particular the gene of resistance to neomycin (neo) under the control of the promoter phosphoglycerate kinase-1 (PGK-1), the expression of the gene of the delta type being suppressed.
According to an advantageous embodiment of the invention, the transgenic mammalian animal or the mammalian cells in which the delta gene is no longer expressed are such that there is replacement of a fragment of the xcex4 gene containing an exon, in particular exon 1, by all or part of a marker gene under the control of a suitable promoter.
The invention also relates to a non-human transgenic mammalian animal or mammalian cells containing the gene of the opiate receptor of the kappa type in which a fragment of the gene of the receptor containing an exon, in particular exon 1, is
either replaced by all or part of a marker gene under the control of a suitable promoter,
or interrupted by the insertion between two contiguous nucleotides of all or part of a marker gene under the control of a suitable promoter, in particular the gene of resistance to neomycin (neo) under the control of the promoter phosphoglycerate kinase-1 (PGK-1), the expression of the kappa gene being suppressed.
According to an advantageous embodiment of the invention, the transgenic mammalian animal or the mammalian cells in which the kappa gene is no longer expressed are such that there is replacement of a fragment of the xcexa gene containing an exon, in particular exon 1, by all or part of a marker gene under the control of a suitable promoter.
The invention also relates to cells cultured from non-human transgenic mammalian animals described above.
According to an advantageous embodiment, the invention relates to cell cultures containing one of the said transgenic constructions.
These cell cultures can be obtained either from cells taken from transgenic animals as defined above or from cell lines using the said transgenic constructions, where this second possibility can be carried out with the aid of standard techniques of cell transfection.
The invention also relates to a non-human transgenic mammal as is obtained by introduction into a blastocyte of embryonal strain cells (ES cells) comprising, in their genome, one of the said transgenic constructions obtained by homologous recombination, selection of chimaeric male animals according to a criterion corresponding to the ES line; crossing of the animals selected with mice, in particular C57 Black 6 mice, to obtain animals which are heterozygous with respect to one of the said constructions, and where appropriate crossing of two heterozygotes to obtain an animal which is homozygous with respect to one of the said constructions.
The homozygote has a 129/C57 Black 6 50/50 genetic base. It is possible to return to a C57 Black 6 genetic base by homozygous crossing with C57 Black 6 mice over at least 12 generations.
C57 Black 6 mice are advantageously chosen since this genetic base is more favourable for certain behaviour experiments.
The invention also relates to a transgenic mammal as produced by crossing transgenic animals which express one of the transgenic constructions defined above.
The invention also relates to a process for obtaining a transgenic model for studying pathologies involving the opiate receptors of the mu type or the opiate receptors of the delta type or the opiate receptors of the kappa type and their treatment, comprising
replacement of the endogenous gene of the opiate receptor of the xcexc type or of the endogenous gene of the opiate receptor delta type or of the endogenous gene of the opiate receptor of the kappa type in cells, in particular embryonal strain (ES) cells of mice, by a construction comprising the gene of the opiate receptor of the mu type or the gene of the opiate receptor of the delta type or the gene of the opiate receptor of the kappa type in which, respectively,
exon 2 of the gene of the opiate receptor of the mu type is interrupted between two contiguous nucleotides by a portion of a marker gene under the control of a suitable promoter, in particular a cassette containing the neo gene under the control of the promoter PGKI or
a fragment containing exon 1 of the gene of the opiate receptor of the delta type is replaced by a marker gene under the control of a suitable promoter, in particular a cassette containing the neo gene under the control of the promoter PGKI, or
a fragment containing exon 1 of the gene of the opiate receptor of the kappa type is replaced by a marker gene under the control of a suitable promoter, in particular a cassette containing the neo gene under the control of the promoter PGKI,
and in particular in which the gene of the opiate receptor of the mu type is interrupted at the BamHI site of exon 2 by insertion of the cassette PGK-neo,
or the genomic fragment SmaIxe2x80x94SmaI of 600 bp containing exon 1 of the gene of the opiate receptor of the delta type is replaced by the cassette PGK-neo,
or the genomic fragment of 235 bp of exon 1 of the gene of the opiate receptor of the kappa type containing the ATG initiator of exon 1 and 232 base pairs downstream of the said ATG is replaced by the cassette PGK-neo, and
introduction of the said cells into embryos, in particular blastocytes of non-human mammals,
selection of male chimaeric animals according to a criterion corresponding to the ES line,
crossing of the animals selected with mice, in particular C57BL/6 mice, to obtain animals which are heterozygous with respect to one of the constructions according to the invention and
where appropriate crossing of two heterozygotes to obtain an animal which is homozygous with respect to one of the constructions according to the invention.
The criterion used is, for example, the color of the hair (agouti).
The invention relates to a process for screening medicaments which act on pathologies involving opiate receptors, in particular the following pathologies: acute or chronic severe pain, toxicomania and prevention or treatment of transplant rejection, comprising:
administration to a transgenic non-human mammal or transgenic non-human mammalian cells containing, instead of the endogenous gene of the opiate receptor of the mu type, or the endogenous gene of the opiate receptor of the delta type, or the endogenous gene of the opiate receptor of the kappa type, a construction containing the gene of the opiate receptor of the mu type, or the gene of the opiate receptor of the delta type, or the gene of the opiate receptor of the kappa type in which, respectively,
exon 2 of the gene of the opiate receptor of the mu type is interrupted between two contiguous nucleotides by a portion of a marker gene under the control of a suitable promoter, in particular a cassette containing the neo gene under the control of the promoter PGKI or
a fragment containing exon 1 of the gene of the opiate receptor of the delta type is replaced by a marker gene under the control of a suitable promoter, in particular a cassette containing the neo gene under the control of the promoter PGKI, or
a fragment containing exon 1 of the gene of the opiate receptor of the kappa type is replaced by a marker gene under the control of a suitable promoter, in particular a cassette containing the neo gene under the control of the promoter PGKI,
and in particular in which the gene of the opiate receptor of the mu type is interrupted at the BamHI site of exon 2 by insertion of the cassette PGK-neo,
or the genomic fragment SmaIxe2x80x94SmaI of 600 bp, containing exon 1 of the gene of the opiate receptor of the delta type is replaced by the cassette neo,
or the genomic fragment of 235 bp of exon 1 of the gene of the opiate receptor of the kappa type containing the ATG initiator of exon 1 and 232 base pairs downstream of the said ATG is replaced by the cassette PGK-neo;
determination of the nociceptive thresholds by the tail immersion and hot plate test after injection of the drugs to be tested,
determination of the response to drugs to be tested by animals in which has been produced chronic pain induced by injection of irritating products, carrageenan and Freund""s adjuvant, and producing monoarthritis or polyarthritis, or the test of sciatic nerve section, or the test of sciatic nerve ligation in the case of neuropathic pain,
or determination of the psychotropic properties of drugs to be tested by the tests of preference of position or of auto-administration, or determination of the level of physical dependence by induction of severe dependence and provocation of withdrawal in the case of toxicomania,
or determination of the mixed lymphocyte reaction and of the life of transplants (Arakawa, K., Akami, T., Okamoto, M., Nakai, I., Oka, T., Nagase, H. Transplantation proceedings (1993) 25:73 8-740) in the case of prevention or treatment of transplant rejection.
The invention also relates to a transgenic construction containing the gene of the opiate receptor of the mu type, or the gene of the opiate receptor of the delta type, or the gene of the opiate receptor of the kappa type in which, respectively,
exon 2 of the gene of the opiate receptor of the mu type is interrupted between two contiguous nucleotides by a portion of a marker gene under the control of a suitable promoter, in particular a cassette containing the neo gene under the control of the promoter PGKI or
a fragment containing exon 1 of the gene of the opiate receptor of the delta type is replaced by a marker gene under the control of a suitable promoter, in particular a cassette containing the neo gene under the control of the promoter PGKI, or
a fragment containing exon 1 of the gene of the opiate receptor of the kappa type is replaced by a marker gene under the control of a suitable promoter, in particular a cassette containing the neo gene under the control of the promoter PGKI,
and in particular in which the gene of the opiate receptor of the mu type is interrupted at the BamHI site of exon 2 by insertion of the cassette neo,
or the genomic fragment SmaIxe2x80x94SmaI of 600 bp containing exon 1 of the gene of the opiate receptor of the delta type is replaced by the cassette PGK-neo,
or the genomic fragment of 235 bp of exon 1 of the gene of the opiate receptor of the kappa type containing the ATG initiator of exon 1 and 232 base pairs downstream of the said ATG is replaced by the cassette PGK-neo.